Un modello Novel di Mild Traumatic Brain Injury per ratti giovani
Summary
The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.
Abstract
Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.
Introduction
Anche se ci sono molti metodi ampiamente utilizzati per la generazione di moderata-grave trauma cranico (TBI), sono state sviluppate poche tecniche per indurre lievi, chiuso ferite alla testa nei roditori. A causa del fatto che il trauma cranico lieve (mTBI) è tre volte più comune di lesione cerebrale moderata e grave combinata 1, è necessario un modello affidabile di mTBI per facilitare la ricerca in materia di fisiopatologia, risultati neurobiologici e comportamentali, e strategie terapeutiche. Ad esempio, gli studi clinici di droga in parte a causa dei limiti di modelli animali attuali 2, negli ultimi dieci anni ci sono stati più di 200 non riusciti per il trattamento di TBI 3. Quando i sistemi di modellazione sono generati per studi di ricerca traslazionale, l'applicabilità dei risultati dipendono dalla validità del modello implementato. Per lo studio della mTBI / sbattimento, un modello animale affidabile non solo imitare il forze biomeccanico responbile per lesioni eziologia, ma potrebbe anche indurre sintomi compatibili con quelli riportati dalla popolazione clinicamente rilevante. Inoltre, perché i bambini sono a rischio particolarmente elevato per mTBI, sistemi di modellazione ottimale sarebbe applicabile ai roditori giovani e giovanili, oltre ai loro equivalenti adulti.
Analisi biomeccaniche di circostanze in cui gli atleti hanno sostenuto mTBIs o lesioni cerebrali concussive indicano che i fattori predittivi più critici per infortunio sono accelerazioni testa rapidi e impatti ad alta velocità 4. La maggior parte dei modelli di roditori attualmente utilizzati per l'induzione di TBI permette poco o nessun movimento della testa 5 (per una rassegna vedi 2). Il modello qui delineato, fornisce un impatto ad alta velocità alla testa di un ratto giovanile fisicamente incontrollato che è accompagnata da una rotazione di 180 ° e caduta libera che applica forze di accelerazione / decelerazione di testa e corpo del soggetto. Tqui sono due vantaggi principali di questa tecnica di peso goccia modificato per l'induzione di mTBI. In primo luogo, il modello produce concussive clinicamente rilevante come sintomatologia senza causare alcun danno evidente al cervello (per la descrizione completa dei risultati comportamentali vedi 6). Coerente anche con i rapporti clinici di sindrome post-concussive, questa tecnica di peso-drop modificato produce risultati eterogenei. Anche se gli effetti della mTBI sono significativi, vi è una sostanziale differenza tra i roditori che hanno subito un mTBI se esaminato su più misure di outcome. In secondo luogo, il metodo consente lo studio di mTBI ripetitivo 7. Poiché la maggior parte dei modelli TBI esistenti infliggere tali lesioni gravi, è spesso difficile indurre una seconda ferita, e quasi impossibile studiare TBI ripetitivo senza danni estesi a tutta la corteccia.
Pertanto, la logica principale per utilizzare la tecnica del peso goccia modificato per l'induction di mTBI è quello di produrre una ferita che rappresenta più da vicino la fisiopatologia e la sintomatologia di concussione e ripetitivo TBI nelle popolazioni giovanili. Con la crescente incidenza di mTBI legati allo sport, cadute, e incidenti automobilistici, soprattutto durante l'infanzia, questo modello roditore unico di mTBI fornisce ai ricercatori uno strumento prezioso per lo studio delle lesioni cerebrali concussive-like che può essere facilmente applicato a multiple-hit paradigmi.
Protocol
Representative Results
Discussion
Reliable modelling systems are needed to effectively cultivate basic science research that has significant translational validity. In response to rising occurrences and popular media, the investigation of mTBI and concussion has become a priority in many disciplines. However, despite increased research, there have been only incremental improvements in therapeutic strategies and treatment options 3. This lack of progress may be partially due to a discrepancy between the modeling systems employed and actual injury etiology. The majority of studies utilized rodent models that failed to reproduce the important biomechanical forces and appropriate post-injury symptomology. The current human definition of mTBI specifies that the injury results from acceleration and deceleration forces associated with a blunt trauma 10. The modified weight drop technique described here is therefore an ideal model for the study of mTBI and concussion because it uses a glancing impact to cause rapid rotational acceleration and deceleration to the head of an unrestrained animal, mimicking the biomechanical forces identified in sports-related injuries and automobile accidents. In addition, this model is easily adapted to examine repetitive mTBI, a phenomena that is emerging as a serious medical and socioeconomic issue. Studies indicate that rodents may be exposed to a series of 10 distinct mTBIs with minimal mortality 7. Finally, the method is inexpensive and can be carried out rapidly, allowing for high-throughput examination of a many therapeutic compounds and treatment regiments.
Just as with any procedural technique, certain aspects of the protocol are particularly important to the generation of reliable results. First, the tin foil needs to be scored effectively. If the tin foil is not properly scored, the force imparted by the weight during the glancing impact will not be enough to propel the juvenile rat through the tin foil onto the collection sponge. In these situations, the rat will remain in the starting position (chest down on the tin foil) and the mTBI will result from the blunt trauma from the weight impacting the stationary head, not the rotational acceleration and deceleration desired. Second, during the induction of the mTBI and the sham injury, the level of anesthetic applied to each rat should be consistent. Owing to the fact that time-to-right is used as marker of mTBI, the researcher should try to ensure that animals receiving a mTBI and animals receiving a sham injury are exposed to similar levels of anesthetic. A major advantage to this technique over many other TBI procedures is the low level and duration of anesthesiology. However, the juvenile rat needs to be non-responsive to a toe or tail pinch to ensure they do not wake-up on the stage before the injury is induced. Finally, in order to maintain a consistent injury etiology, the positioning of the rat’s head is particularly important. Ideally the weight should impact the center of the dorsal surface of the head. Caution should be taken to avoid positioning the path of the weight too near the caudal/posterior portion of the head, as impacting the brainstem and cerebellum is associated with increased mortality and seizure activity.
Based upon the biomechanical pathophysiology of injury induction and the behavioural outcomes examined, the modified weight-drop technique appears to be a reliable model for the investigation of paediatric mTBI and concussion. Although preliminary studies of this novel model have assessed some basic molecular and structural changes 7 future studies will be needed to ascertain how the brain responds to a mTBI with this injury etiology. An in-depth analysis of the neuroanatomical and biological changes that occur at the cellular and epigenetic level would increase model validity and translational applicability. In addition to stimulating the generation of targeted pharmacological therapies, understanding the pathophysiological changes that occur in the brain in response to mTBI and concussion would also direct the research related to clinical biomarkers that have the ability to predict outcomes following injury.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Irene Ma, Rose Tobais, and Jong Rho for their technical assistance. Funding was provided to MJE by the Department of Pediatrics at the University of Calgary, the Alberta Children’s Hospital Foundation (ACHF) and the Alberta Children’s Hospital Research Institute (ACHRI). The Postdoctoral fellowship for RM was provided by ACHF.
Materials
| Brass Weights | Ginsberg Scientific | 7-2500-2 | Need to have metal loop attached to base |
| Alluminum Foil | Alcan | Available at most grocery stores | |
| Masking Tape | Commercially available | ||
| U-Shaped Plastic Stand | Constructed by Laboratory | ||
| Clamp Stand | Sigma-Aldrich | Z190357 | |
| Plastic Guide Tube | Could be constructed or purchased at a hardware store | ||
| Fishing Line | Angler 10lb | Purchased from a sporting goods retailer | |
| Isoflurane | Pharmaceutical Partners of Canada | DIN 02237518 | Inhalation Anesthetic |
| Topical Lidocaine (30ml) | Astra Zeneca | DIN 0001694 | Xylocaine Jelly 2% |
| Cotton Swabs | Commercially available | ||
| Heating Pad – 3 heat setting | Commercially available | ||
| Stop Watch | Sportline | L303 | Purchased from a sporting goods retailer |
| Video Camera | Sony | HDR-CX260V | |
| Sprague Dawley Rats | Charles River Laboratories | SAS SD 40 | Male and females ordered from Charles River Laboratories and pups bred in-house |
| Balance Beam | Constructed by Laboratory |
Riferimenti
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- Mychasiuk, R., Farran, A., Esser, M. J. Assessment of an experimental rodent model of pediatric mild traumatic brain injury. Journal of Neurotrauma. 31 (8), (2014).
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- Mychasiuk, R., Hehar, H., Farran, A., Esser, M. J. Mean Girls: Sex differences in the effects of mild traumatic brain injury on the social dynamics of juvenile rat play behaviour. Behavioural Brain Research. 259, (2014).
- Centers for Disease Control and Prevention. . Report to congress on mild traumatic brain injury in the United States: Steps to prevent a serious public health problem. , 1-47 (2003).
